Biomedical subjects

Tetsuya Kawakita

Publications and source records attributed to Tetsuya Kawakita.

At least 19 recordsLinked to original sources

PubMed2006

Comparison of deep lamellar keratoplasty and penetrating keratoplasty for lattice and macular corneal dystrophies.

PURPOSE: To compare the therapeutic outcomes after deep lamellar keratoplasty (DLKP) and penetrating keratoplasty (PKP) in patients with lattice corneal dystrophy (LCD) and macular corneal dystrophy (MCD). DESIGN: Age-matched control study. METHODS: We reviewed the clinical records of 84 eyes with LCD or MCD who had DLKP (41 eyes) or PKP (43 eyes). Primary pathology consisted of 60 eyes with LCD and 24 eyes with MCD. DLKP was performed by either removing stromal tissue gradually, or by viscodissection of Descemet's membrane. Graft clarity, best-corrected visual acuity (BCVA), endothelial density, and complications were compared between DLKP and PKP, as well as between LCD and MCD. RESULTS: All 84 eyes showed a postoperative improvement in visual acuity. The median final BCVA was not significantly different between PKP and DLKP groups. Endothelial cell loss rates were similar for DLKP and PKP. While the MCD-DLKP group showed progressive decrease in endothelial density, this was not observed in the LCD-DLKP group after surgery. In the DLKP group, most of the complications occurred intraoperatively or in the early phase, whereas late phase complications such as endothelial rejection and secondary glaucoma were the main complications in the PKP group. CONCLUSIONS: PKP is no longer an automatic choice for the surgical treatment for LCD and MCD; DLKP seems to be a safe alternative. While DLKP is a favorable method for LCD, MCD may not be a good candidate, as it might show progressive decrease in the corneal endothelium postoperatively.

Adult↗

PubMed2006

Accessory cell populations in the cornea.

According to the "classical" doctrine, resident cells of the cornea include the stratified epithelial cells, quiescent keratocytes, and a single layer of nondividing endothelial cells. However, it has become increasingly evident that other cell types are involved in the homeostasis of the cornea. The presence of various cell types from different lineages has raised concern among researchers as to what we are actually "seeing" in the cornea. Although definitive conclusions cannot yet be drawn, this review attempts to clarify the various accessory cell types reported in the human and murine cornea. The epithelial layer of the limbal area includes melanocytes, as well as antigen-presenting cells that are also present in the peripheral clear cornea. The most debated tissue currently is perhaps the corneal stroma, where resident keratocytes are not as large a population as was previously believed. Bone marrow-derived cells are found in the cornea, and these may not express the typical HLA molecules usually found on the surface of antigen-presenting cells. Nerve fibers extend though the superficial stroma to form a plexus beneath the epithelium. Although the cell body of the neuron itself is not in the cornea, neural glial cells, such as Schwann cells, are present in the stroma. The use of specific molecular markers and high-quality imaging techniques will be required to fully elucidate the various accessory cells of the cornea and their function.

Animals↗

PubMed2006

Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane.

PURPOSE: Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS: The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS: In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached approximately 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 microM LY294002 or 50 microM SR13668 and was significantly suppressed by 10 microM U0126, but was not affected by 10 microM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS: Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.

Amnion↗

PubMed2006

Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium.

PURPOSE: To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. METHODS: Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. RESULTS: In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. CONCLUSIONS: The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.

Adenoviridae↗

PubMed2006

Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane.

PURPOSE: To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS: Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin alpha6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS: All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS: After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.

Amnion↗

PubMed2006

Cytokeratin 15 can be used to identify the limbal phenotype in normal and diseased ocular surfaces.

PURPOSE: To elucidate the expression pattern of K15, K19, K14, and K12 in human and mouse ocular surface epithelium as putative markers of epithelial phenotype. METHODS: Immunohistochemical staining with specific antibodies for K15, K19, K14, and K12 was performed in human donor cornea tissue and normal ICR mouse corneas, with emphasis on localization of immunopositive cells. Immunohistochemistry was performed in a limbus-deficient mouse model as well as in clinical samples of pannus surgically removed from a thermal burn and a patient with Saltzmann's dystrophy. Staining patterns were classified as limited to the most basal layer (K(bas)), basal and suprabasal layers (K(bas-sup)), predominantly in suprabasal layers (K(sup)) and negative staining (K(-)). RESULTS: In human conjunctival epithelium, strong expression of K15 was observed in basal cells, whereas K19 was expressed in both basal and suprabasal layers (K15(bas)/K19(bas-sup)/K12(-)). Limbal epithelial cells were K15(bas-sup)/K19(bas-sup)/K12(sup), whereas epithelial cells in the central cornea were K15(-)/K19(bas-sup)/K12(bas-sup). In contrast, the mouse ocular surface demonstrated a different expression pattern of K15 and K19 than did the human tissue in the conjunctiva (K15(bas-sup)/K19(bas)/K12(-)) and the limbus (K15(bas-sup)/K19(bas)/K12(sup)). Neither K15 nor K19 was expressed in the central mouse cornea (K15(-)/K19(-)/K12(bas-sup)). Similar cytokeratin expression was observed in conjunctivalized corneas in mice and in surgically removed pannus tissue. CONCLUSIONS: Although the expression of K15 and K19 differ in humans and mice, specific staining patterns can be used to characterize the epithelial phenotype in normal and diseased ocular surface.

Animals↗

PubMed2005

Amniotic membrane induces apoptosis of interferon-gamma activated macrophages in vitro.

Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-gamma (IFN-gamma). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-alpha) concentration. Lysates of cells were subjected to Western blot analyses of IKK-alpha, IKK-beta, p65 (RelA) subunit of nuclear factor kappaB (NF-kappaB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-gamma and on AM without IFN-gamma. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-gamma activation, and this phenomenon became apparent only after 48 hr. IFN-gamma-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-alpha during 72 hr culturing. In contrast, there was no NO and TNF-alpha production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-gamma-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-alpha, IKK-beta, p65 (RelA) subunit of NF-kappaB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-gamma-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-gamma activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-kappaB and Akt-FKHR signalling pathways.

Amnion↗

PubMed2005

Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-beta signaling.

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-beta2 and TGF-beta receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express alpha-smooth muscle actin, even when 10 ng/ml TGF-beta1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-beta signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-beta. Collectively, these results indicate that down-regulation of Smad-mediated TGF-beta signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-beta signaling, achieved by AM stromal matrix in part via suppression of TGF-beta gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Animals↗

PubMed2005

Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture.

The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.

Amnion↗

PubMed2005

TGF-beta1, IL-1beta, and Th2 cytokines stimulate vascular endothelial growth factor production from conjunctival fibroblasts.

Giant papillary formation containing newly formed vessels is a major characteristic of severe allergic conjunctivitis, such as atopic keratoconjunctivitis (AKC) or vernal keratoconjunctivitis (VKC). We examined production of vascular endothelial growth factor (VEGF) from cultured conjunctival fibroblasts from normal volunteers under stimulation with type 1-, type 2-helper T cell derived and proinflammatory cytokines to investigate the mechanism of giant papillae formation in AKC/VKC. Primary cultured conjunctival fibroblasts were incubated with interleukin (IL)-4, IL-13, IL-1beta, IL-2, tumor necrotizing factor (TNF)-alpha, interferon (IFN)-gamma, or transforming growth factor (TGF)-beta1. Effects of cytokines on VEGF protein secretion in supernatant were assessed by ELISA, and VEGF mRNA expression in cultured cells were assessed by quantitative PCR. TGF-beta1 most effectively increased VEGF concentration with dose- and time-dependent manner IL-1beta, IL-4, and IL-13 significantly increased VEGF concentration. Though IL-2 also showed slight increase of VEGF concentration, it was not statistically significant. TNF-alpha and INF-gamma did not increase VEGF concentration. Quantitative PCR showed significant increase of VEGF mRNA in TGF-beta1, IL-1beta, and IL-4 stimulated fibroblasts. TGF-beta1, IL-1beta, and Th2 cytokines from allergic inflammatory cells induced VEGF production in conjunctival fibroblasts, and may play a crucial role in neovascularization and formation of giant papillae in AKC/VKC.

Cell Division↗

PubMed2005

Correlation of corneal complications with eyelid cicatricial pathologies in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis syndrome.

PURPOSE: To look at the correlation between many factors (time of hospitalization, floppy eyelid syndrome, trichiasis, open lacrimal puncta, symblepharon, and aqueous tear deficiency) and corneal complications in Stevens-Johnson syndrome (SJS). DESIGN: Observational cases series. PATIENTS: Clinical data were retrospectively reviewed from 38 patients (32.7+/-20.1 years old) with SJS (n = 11) and with toxic epidermal necrolysis (TENS) (n = 27) from January 2002 to August 2004. One case report with SJS was included to verify the presence of tarsal/lid margin ulceration at the acute stage. METHODS: The medical history was retrieved regarding presumed causative medications used within 15 days and the duration of hospitalization. Data of the latest photographic documentation and eye examination were compared and correlated in a masked fashion. MAIN OUTCOME MEASURES: Floppy eyelid, trichiasis, lid margin keratinization, meibomian gland orifice metaplasia, symblepharon, tarsal scar, and corneal complications. RESULTS: Acute SJS/TENS was characterized by tarsal conjunctival ulceration. Keratinization of the eyelid margin with variable degrees of meibomian gland dysfunction was observed in all cases. Floppy eyelid, trichiasis, partially or totally opened lacrimal punctum, symblepharon, and aqueous tear deficiency were not significantly correlated with corneal complications. In contrast, there was a strong correlation between the severity of eyelid margin and tarsal pathology and the extent of corneal complications (Spearman r, 0.54; P = 0.0005). A multivariable regression analysis also showed that the extent of eyelid and tarsal pathology had a significant effect on corneal complications (coefficient, 0.84; P = 0.006). CONCLUSIONS: Patients with acute SJS/TENS are characterized by severe inflammation and ulceration of the tarsal conjunctiva and lid margins. If left unattended, lid margin keratinization and tarsal scar, together with lipid tear deficiency, contribute to corneal complications because of blink-related microtrauma. Attempts to suppress inflammation and scarring by amniotic membrane transplantation at the acute stage and to prevent microtrauma at the chronic stage are vital to avoid sight-threatening complications.

Adolescent↗

PubMed2005

Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure.

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.

Air↗

PubMed2005

Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.

PURPOSE: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement. METHODS: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model. RESULTS: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice. CONCLUSIONS: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Amnion↗

PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗

PubMed2005

High prevalence of Demodex in eyelashes with cylindrical dandruff.

PURPOSE: To determine the prevalence of Demodex in eyelashes with cylindrical dandruff (CD). METHODS: A modified sampling and counting method was applied to 55 clinical cases. Patients were divided in to group A (n = 20) with diffuse CD, group B (n = 12) with sporadic CD, and group C (n = 23) with clean lashes or greasy scales, of which the latter was divided into subgroup C1 (n = 15) without lid hygiene and subgroup C2 (n = 8) using daily lid hygiene for the past year. Each patient underwent a routine complete eye examination and modified counts of Demodex. RESULTS: Demodex was found in all group A and B patients (n = 32) with CD, which was significantly higher than the 22% of group C patients (n = 23) without CD (P < 0.001). The Demodex counts were 4.1 +/- 1.0 and 2.0 +/- 1.2 per epilated lash with retained CD, significantly higher than the 0.2 +/- 0.5 and 0.2 +/- 0.4 per lash without retained CD in groups A and B, respectively (each P < 0.001) and than the 0.01 +/- 0.09 and 0.12 +/- 0.41 per lash in subgroups C1 and C2, respectively (each P < 0.001). Demodex was still found in CD fragments left on the lid skin after epilation. Five Demodex brevis mites were found among the 422 Demodex specimens. CONCLUSIONS: The modified sampling and counting method showed that the prior controversy regarding Demodex has resulted from miscounting and confirmed that lashes with CD are pathognomonic for ocular Demodex infestation. Lid hygiene with shampoo reduces Demodex counts but does not eradicate the mites.

Adult↗

PubMed2004

How does amniotic membrane work?

Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine.

Journal Article↗

PubMed2004

Quantitative evaluation of eyelid elasticity using the cutometer SEM575 and its clinical application in assessing the efficacy of tacrolimus ointment treatment in eyelid atopic dermatitis.

PURPOSE: To study the clinical efficacy of a noninvasive suction device that measures eyelid skin elasticity in the treatment of eyelid atopic dermatitis using tacrolimus (FK-506) ointment. METHODS: Ten patients with eyelid atopic dermatitis treated with tacrolimus ointment and 10 normal volunteers participated in this study. The cutometer SEM575 was used to quantitatively evaluate eyelid skin elasticity. Severity of the eyelid atopic dermatitis was scored, and comparisons were made before and after treatment. RESULTS: Skin elasticity of patients with eyelid atopic dermatitis was significantly lower than that of normal volunteers (31.3 +/- 5.2% vs 40.2 +/- 7.8%, respectively). Skin elasticity of patients with eyelid atopic dermatitis improved significantly to 37.5 +/- 6.3% after treatment with tacrolimus ointment. The total severity score for eyelid atopic dermatitis also improved from 2.77 +/- 1.11 to 1.77 +/- 1.15. CONCLUSION: Measurement of skin elasticity using the cutometer SEM575 is a useful and reliable method for objective and quantitative evaluation of eyelid skin condition in patients with eyelid atopic dermatitis. The efficacy of short-term tacrolimus ointment treatment in patients with eyelid atopic dermatitis was confirmed quantitatively using this apparatus.

Adult↗

PubMed2004

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

PURPOSE: To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. METHODS: The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. RESULTS: Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. CONCLUSIONS: The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker.

Adult↗